Integration of multiple downstream signals determines the net effect of insulin on MAP kinase vs. PI 3′-kinase activation: potential role of insulin-stimulated H 2O 2

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Cellular insulin stimulation generates a burst of H 2O 2 that modulates protein-tyrosine phosphorylation in the insulin action pathway, in part by the inhibition of redox-sensitive protein-tyrosine phosphatases [J. Biol. Chem. 276 (2001) 21938]. Blocking the insulin-induced rise in H 2O 2 with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) strongly attenuated the activation of phosphatidylinositol 3′ (PI 3′)-kinase, Akt and GLUT4 translocation by insulin in 3T3-L1 adipocytes; however, under identical conditions, we observed a paradoxical increase in the activation of p42/p44 mitogen-activated protein (MAP) kinase. DPI inhibited the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1/2, and also reduced the association of Grb2 with IRS-1, suggesting that the effect of DPI on MAP kinase activation occurred downstream of the IR and IRS proteins. DPI increased the insulin-stimulated phosphorylation of p42/p44 MAP kinase with no change in basal, and increased insulin-stimulated MAP kinase kinase (MEK) activity by a similar degree. DPI enhanced basal Grb2–Sos binding and reduced the effect of insulin to potentiate the dissociation of the Grb2–Sos complex, suggesting that the effect of DPI was mediated upstream of Raf-1. Cell treatment with dibutyryl cAMP significantly reduced the enhancement of MAP kinase activation in the presence of DPI. However, forskolin, acting in a PKA-independent manner, increased the insulin stimulation of MAP kinase and MEK, but fully abrogated the effect of DPI to enhance these insulin responses. PLCγ inhibition with U73122 blocked the insulin stimulation of MAP kinase and MEK as well as the enhancing effect of DPI on these responses. PKC activation strongly stimulated MAP kinase and MEK activation, even in the presence of U73122, consistent with PKC acting downstream of PLCγ. These data show that the insulin-stimulated oxidant signal differentially affects the two major downstream components of the insulin signaling pathway, PI 3′-kinase and MAP kinase, and cross-talk between insulin action, PLCγ and, to a lesser extent, PKA modulates the net cellular effects of insulin-stimulated cellular H 2O 2.